Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.24 (mitogen-activated protein kinase)
 95,810 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Huntingtin (Htt) is a large protein of 3144 amino acids, whose function and regulation have not been well defined. Polyglutamine (polyQ) expansion in the N terminus of Htt causes the neurodegenerative disorder Huntington disease (HD). The cytotoxicity of mutant Htt is modulated by proteolytic cleavage with caspases and calpains generating N-terminal polyQ-containing fragments. We hypothesized that phosphorylation of Htt may modulate cleavage and cytotoxicity. In the present study, we have mapped the major phosphorylation sites of Htt using cell culture models (293T and PC12 cells) expressing full-length myc-tagged Htt constructs containing 23Q or 148Q repeats. Purified myc-tagged Htt was subjected to mass spectrometric analysis including matrix-assisted laser desorption/ionization mass spectrometry and nano-HPLC tandem mass spectrometry, used in conjunction with on-target alkaline phosphatase and protease digestions. We have identified more than six novel serine phosphorylation sites within Htt, one of which lies in the proteolytic susceptibility domain. Three of the sites have the consensus sequence for ERK1 phosphorylation, and addition of ERK1 inhibitor blocks phosphorylation at those sites. Other observed phosphorylation sites are possibly substrates for CDK5/CDC2 kinases. Mutation of amino acid Ser-536, which is located in the proteolytic susceptibility domain, to aspartic acid, inhibited calpain cleavage and reduced mutant Htt toxicity. The results presented here represent the first detailed mapping of the phosphorylation sites in full-length Htt. Dissection of phosphorylation modifications in Htt may provide clues to Huntington disease pathogenesis and targets for therapeutic development.
 ...
PMID:Huntingtin phosphorylation sites mapped by mass spectrometry. Modulation of cleavage and toxicity. 1678 7

MSK1 (mitogen- and stress-activated kinase 1) is a dual kinase domain protein that acts downstream of the ERK1/2 (extracellular-signal-regulated kinase 1/2) and p38 MAPK (mitogen-activated protein kinase) signalling pathways in cells. MSK1, and its related isoform MSK2, phosphorylate the transcription factors CREB (cAMP-response-element-binding protein) and ATF1 (activating transcription factor 1), and the chromatin proteins histone H3 and HMGN1 (high-mobility-group nucleosomal-binding protein 1) in response to either mitogenic stimulation or cellular stress. MSK1 activity is tightly regulated in cells, and activation requires the phosphorylation of MSK1 by either ERK1/2 or p38a. This results in activation of the C-terminal kinase domain, which then phosphorylates further sites in MSK1, leading to the activation of the N-terminal kinase domain and phosphorylation of substrates. Here, we use precursor ion scanning MS to identify five previously unknown sites in MSK1: Thr630, Ser647, Ser657, Ser695 and Thr700. One of these sites, Thr700, was found to be a third site in MSK1 phosphorylated by the upstream kinases ERK1/2 and p38a. Mutation of Thr700 resulted in an increased basal activity of MSK1, but this could be further increased by stimulation with PMA or UV-C radiation. Surprisingly, however, mutation of Thr700 resulted in a dramatic loss of Thr581 phosphorylation, a site essential for activity. Mutation of Thr700 and Thr581 to an alanine residue resulted in an inactive kinase, while mutation of both sites to an aspartic acid residue resulted in a kinase with a significant basal activity that could not be further stimulated. Together these results are consistent with a mechanism by which Thr700 phosphorylation relieves the inhibition of MSK1 by a C-terminal autoinhibitory helix and helps induce a conformational shift that protects Thr581 from dephosphorylation.
 ...
PMID:Identification of novel phosphorylation sites in MSK1 by precursor ion scanning MS. 1711 22

Previous studies have suggested that Mcl-1, an antiapoptotic Bcl-2 homolog that does not exhibit appreciable affinity for the caspase 8-generated C-terminal Bid fragment (tBid), diminishes sensitivity to tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL). This study was performed to determine the mechanism by which Mcl-1 confers TRAIL resistance and to evaluate methods for overcoming this resistance. Affinity purification/immunoblotting assays using K562 human leukemia cells, which contain Mcl-1 and Bcl-x(L) as the predominant antiapoptotic Bcl-2 homologs, demonstrated that TRAIL treatment resulted in binding of tBid to Bcl-x(L) but not Mcl-1. In contrast, TRAIL caused increased binding between Mcl-1 and Bak that was diminished by treatment with the caspase 8 inhibitor N-(N(alpha)-acetylisoleucylglutamylthreonyl) aspartic acid (O-methyl ester)-fluoromethyl ketone (IETD(OMe)-fmk) or the c-Jun N-terminal kinase inhibitor SP600125. In addition, TRAIL caused increased binding of Bim and Puma to Mcl-1 that was inhibited by IETD(OMe)-fmk but not SP600125. Further experiments demonstrated that down-regulation of Mcl-1 by short hairpin RNA or the kinase inhibitor sorafenib increased TRAIL-induced Bak activation and death ligand-induced apoptosis in a wide variety of neoplastic cell lines as well as clinical acute myelogenous leukemia specimens. Collectively, these observations not only suggest a model in which Mcl-1 confers TRAIL resistance by serving as a buffer for Bak, Bim, and Puma, but also identify sorafenib as a potential modulator of TRAIL sensitivity.
 ...
PMID:Mcl-1 as a buffer for proapoptotic Bcl-2 family members during TRAIL-induced apoptosis: a mechanistic basis for sorafenib (Bay 43-9006)-induced TRAIL sensitization. 1769 40

Recent work indicates that mitogen-activated protein kinase kinase (MEK)1 signaling at the G2/M cell cycle transition unlinks the contiguous mammalian Golgi apparatus and that this regulates cell cycle progression. Here, we sought to determine the role in this pathway of Golgi reassembly protein (GRASP)55, a Golgi-localized target of MEK/extracellular signal-regulated kinase (ERK) phosphorylation at mitosis. In support of the hypothesis that GRASP55 is inhibited in late G2 phase, causing unlinking of the Golgi ribbon, we found that HeLa cells depleted of GRASP55 show a fragmented Golgi similar to control cells arrested in G2 phase. In the absence of GRASP55, Golgi stack length is shortened but Golgi stacking, compartmentalization, and transport seem normal. Absence of GRASP55 was also sufficient to suppress the requirement for MEK1 in the G2/M transition, a requirement that we previously found depends on an intact Golgi ribbon. Furthermore, mimicking mitotic phosphorylation of GRASP55 by using aspartic acid substitutions is sufficient to unlink the Golgi apparatus in a gene replacement assay. Our results implicate MEK1/ERK regulation of GRASP55-mediated Golgi linking as a control point in cell cycle progression.
 ...
PMID:GRASP55 regulates Golgi ribbon formation. 1843 98

We investigated the induction of apoptosis via deamidation of Bcl-xL and translocation of Bax to the mitochondria by treatment with GSH-DXR. GSH-DXR treatment of HepG2 cells, which did not express GST P1-1, exhibited deamidation of Bcl-xL, and the degree of deamidation was related to the activation of caspase-3. Overexpression of GST P1-1 in HepG2 cells decreased both the Bcl-xL deamidation and caspase-3 activation induced by treatment with GSH-DXR. Bcl-xL deamidation and caspase-3 activation were also suppressed by co-treatment with SP600125, a specific inhibitor of JNK activity. Overexpression of wild-type Bcl-xL in HepG2 decreased GSH-DXR-induced apoptosis although deamidation was observed. However, expression of the deamidated mutant of Bcl-xL, in which aspartic acid was substituted for both arginine 52 and 66 (N52,66D-Bcl-xL), exhibited high sensitivity for the induction of apoptosis. Expression of the Bcl-xL mutant, in which alanine was substituted for both arginine 52 and 66 (N52,66A-Bcl-xL), suppressed deamidation and showed resistance to the induction of apoptosis by treatment with GSH-DXR. On the other hand, endogenous Bax and overexpressed Flag-Bax were localized in the cytosolic fraction of HepG2 cells. Treatment of the cells with GSH-DXR caused translocation of Flag-Bax to the mitochondrial fraction following the induction of apoptosis. The induced apoptosis was enhanced by the expression of Flag-Bax. Moreover, Flag-Bax was partly located in the mitochondrial fraction in N52,66D-Bcl-xL-expressed cells without the induction of apoptosis. Therefore, the induction of apoptosis by treatment of HepG2 with GSH-DXR was enhanced, thereby facilitating the release of cytochrome c by both deamidated inactivation of Bcl-xL and functional translocation of Bax to the mitochondria via JNK activation. Deamidation of Bcl-xL might be induced in order to translocate Bax to the mitochondria.
 ...
PMID:The association of deamidation of Bcl-xL and translocation of Bax to the mitochondria through activation of JNK in the induction of apoptosis by treatment with GSH-conjugated DXR. 1863 61

It is known that activity modulates neuronal differentiation in the adult brain but the signalling mechanisms underlying this process remain to be identified. We show here that activity requires soluble amyloid precursor protein (sAPP) to enhance neurite outgrowth of young neurons differentiating from neural stem cells. Inhibition of sAPP secretion and anti-APP antibodies both abolished the effect of depolarization on neurite outgrowth, whereas exogenous sAPPalpha, similar to depolarization, induced neurite elongation. Depolarization and sAPPalpha both required active N-methyl-D-aspartic acid receptor (NMDAR) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) recruitment to induce neurite outgrowth. However, depolarization and sAPPalpha played different roles in modulating this signalling cascade. Depolarization induced ERK phosphorylation with fast kinetics via activation of NMDAR. By contrast, acute application of sAPPalpha did not lead to ERK activation. However, continuous generation of sAPPalpha was necessary for depolarization-induced ERK phosphorylation, indicating that sAPPalpha promotes MAPK/ERK recruitment by an indirect mechanism. In addition, we found that blockade of NMDAR down-regulated APP expression, whereas depolarization increased sAPPalpha, suggesting that activity may also act upstream of sAPP signalling by regulating the amount of cellular APP and extracellular sAPPalpha. Finally, we show that soluble amyloid precursor-like protein 2 (sAPLP2), but not sAPLP1, is functionally redundant to sAPP in promoting neurite outgrowth and that soluble members of the APP family require membrane-bound APP to enhance neurite outgrowth. In summary, these experiments indicate a novel role of APP family members in activity-dependent neuronal differentiation.
 ...
PMID:Activity requires soluble amyloid precursor protein alpha to promote neurite outgrowth in neural stem cell-derived neurons via activation of the MAPK pathway. 1871 33

Bone sialoprotein (BSP) is an abundant protein in the extracellular matrix of bone that has been suggested to have several different physiological functions, including the nucleation of hydroxyapatite (HA), promotion of cell attachment and binding of collagen. Studies in our lab have demonstrated that increased expression of BSP in osteoblast cells can increase expression of the osteoblast-related genes Runx2 and Osx as well as alkaline phosphatase and osteocalcin and increase matrix mineralization. To determine the molecular mechanisms responsible for the BSP-mediated increase in osteoblastic differentiation, several functional domain mutants of BSP were expressed in primary rat bone osteoblastic cells, including the contiguous glutamic acid sequences (polyGlu) and the arginine-glycine-aspartic acid (RGD) motif. Markers of osteoblast differentiation, including matrix mineralization and alkaline phosphatase staining, were increased in cells expressing BSP mutants of the polyGlu sequences but not in cells expressing RGD-mutated BSP. We also determined the dependence on integrin-associated pathways in promoting BSP-mediated differentiation responses in osteoblasts by demonstrating the activation of focal adhesion kinase, MAP kinase-associated proteins ERK1/2, ribosomal s6 kinase 2 and the AP-1 protein cFos. Thus, the mechanism regulating osteoblast differentiation by BSP was determined to be dependent on integrin-mediated intracellular signaling pathways.
 ...
PMID:Activation of the mitogen-activated protein kinase pathway by bone sialoprotein regulates osteoblast differentiation. 1872 50

Tolerance to otherwise lethal cerebral ischemia in vivo or to oxygen-glucose deprivation (OGD) in vitro can be induced by prior transient exposure to N-methyl-D-aspartic acid (NMDA): preconditioning in this manner activates extrasynaptic and synaptic NMDA receptors and can require bringing neurons to the "brink of death." We considered if this stressful requirement could be minimized by the stimulation of primarily synaptic NMDA receptors. Subjecting cultured cortical neurons to prolonged elevations in electrical activity induced tolerance to OGD. Specifically, exposing cultures to a K(+)-channel blocker, 4-aminopyridine (20-2500 microm), and a GABA(A) receptor antagonist, bicuculline (50 microm) (4-AP/bic), for 1-2 days resulted in potent tolerance to normally lethal OGD applied up to 3 days later. Preconditioning induced phosphorylation of ERK1/2 and CREB which, along with Ca(2+) spiking and OGD tolerance, was eliminated by tetrodotoxin. Antagonists of NMDA receptors or L-type voltage-gated Ca(2+) channels (L-VGCCs) applied during preconditioning decreased Ca(2+) spiking, phosphorylation of ERK1/2 and CREB, and OGD tolerance more effectively when combined, particularly at the lowest 4-AP concentration. Inhibiting ERK1/2 or Ca(2+)/calmodulin-dependent protein kinases (CaMKs) also reduced Ca(2+) spiking and OGD tolerance. Preconditioning resulted in altered neuronal excitability for up to 3 days following 4-AP/bic washout, based on field potential recordings obtained from neurons cultured on 64-channel multielectrode arrays. Taken together, the data are consistent with action potential-driven co-activation of primarily synaptic NMDA receptors and L-VGCCs, resulting in parallel phosphorylation of ERK1/2 and CREB and involvement of CaMKs, culminating in a potent, prolonged but reversible, OGD-tolerant phenotype.
 ...
PMID:Elevated synaptic activity preconditions neurons against an in vitro model of ischemia. 1884 40

Glutamate and norepinephrine (NE) are believed to mediate the long-lasting synaptic plasticity in the accessory olfactory bulb (AOB) that underlies pheromone recognition memory. The mechanisms by which these neurotransmitters bring about the synaptic changes are not clearly understood. In order to study signals that mediate synaptic plasticity in the AOB, we used AOB neurons in primary culture as a model system. Because induction of pheromone memory requires coincident glutamatergic and noradrenergic input to the AOB, and requires new protein synthesis, we reasoned that glutamate and NE must induce gene expression in the AOB. We used a combination of agonists that stimulate alpha1 and alpha2 adrenergic receptors in combination with N-methyl-d-aspartic acid and tested expression of the immediate-early gene (IEG) c-Fos. We found that the glutamatergic and noradrenergic stimulation caused significant induction of c-Fos mRNA and protein. Induction of c-Fos was significantly reduced in the presence of inhibitors of protein kinase C, mitogen-activated protein kinase (MAPK) and phospholipase C. These results suggest that glutamate and NE induce gene expression in the AOB through a signaling pathway mediated by protein kinase C and MAPK.
 ...
PMID:Signal transduction and gene expression in cultured accessory olfactory bulb neurons. 1884 4

MAP2Ks are dual-specificity protein kinases functioning at the center of three-tiered MAP kinase modules. The structure of the kinase domain of the MAP2K MEK6 with phosphorylation site mimetic aspartic acid mutations (MEK6/DeltaN/DD) has been solved at 2.3 angstroms resolution. The structure reveals an autoinhibited elongated ellipsoidal dimer. The enzyme adopts an inactive conformation, based upon structural queues, despite the phosphomimetic mutations. Gel filtration and small-angle X-ray scattering analysis confirm that the crystallographically observed ellipsoidal dimer is a feature of MEK6/DeltaN/DD and full-length unphosphorylated wild-type MEK6 in solution. The interface includes the phosphate binding ribbon of each subunit, part of the activation loop, and a rare "arginine stack" between symmetry-related arginine residues in the N-terminal lobe. The autoinhibited structure likely confers specificity on active MAP2Ks. The dimer may also serve the function in unphosphorylated MEK6 of preventing activation loop phosphorylation by inappropriate kinases.
 ...
PMID:The structure of the MAP2K MEK6 reveals an autoinhibitory dimer. 1914 Dec 86


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>